Higher eukaryotes have many distinct esterases. Among the different types of esterases are those that act on carboxylic esters, also known as carboxylesterases. Carboxylesterases have been classified into three categories (A, B and C) on the basis of differential patters of inhibition by organophosphates (Myers et al. (1988) Mol. Biol. Evol. 5(2):113-119). Sequence analysis of a number of type-B carboxylesterase demonstrates their evolutionary interrelatedness. Members of the type B carboxylesterase family include acetylcholincarboxylesterases, mammalian cholincarboxylesterases, thyroglobulin, neuroligins, and mammalian bile salt activated lipases.
The type B family of carboxylesterase also includes vitamin K-dependent carboxylases. Vitamin K-dependent gamma-glutamyl carboxylases catalyze the posttranslational conversion of glutamic acid to gamma-carboxyglutamic acid, an amino acid critical to the function of the vitamin K-dependent blood coagulation proteins (Begley et al. (2000) J. Biol. Chem. 275:36245-36249). Incomplete gamma carboxylation of blood clotting factors is associated with poor coagulation.
For the gamma carboxylation event to occur, both vitamin K and the presence of a gamma carboxylation recognition site on the substrate are required. Gamma carboxyglutamic acid confers calcium binding ability upon the modified protein. For blood clotting factors, calcium binding results in a conformational change that exposes hydrophobic residues for interactions with membranes.
Although gamma carboxylation was a biochemical event first characterized in the mammalian blood clotting cascade, it has been found to have a more generalized applicability. For example, vitamin K-dependent gamma carboxylation of glutamate residues has also been detected for a variety of other proteins including bone proteins, PRGP1, PRGP2, and neuropeptides (Walker et al. (2000) J. Biol. Chem., December 7 epub ahead of print).
The present invention is based, in part, on the discovery of a novel carboxylesterase family member, referred to herein as xe2x80x9c53010.xe2x80x9d The nucleotide sequence of a cDNA encoding 53010 is shown in SEQ ID NO:1, and the amino acid sequence of a 53010 polypeptide is shown in SEQ ID NO:2. In addition, the nucleotide sequences of the coding region are depicted in SEQ ID NO:3.
Accordingly, in one aspect, the invention features a nucleic acid molecule that encodes a 53010 protein or polypeptide, e.g., a biologically active portion of the 53010 protein. In a preferred embodiment the isolated nucleic acid molecule encodes a polypeptide having the amino acid sequence of SEQ ID NO:2. In other embodiments, the invention provides isolated 53010 nucleic acid molecules having the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, a full complement of SEQ ID NO:1 or SEQ ID NO:3. In still other embodiments, the invention provides nucleic acid molecules that are substantially identical (e.g., naturally occurring allelic variants) to the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3. In other embodiments, the invention provides a nucleic acid molecule which hybridizes under a stringency condition described herein to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, wherein the nucleic acid encodes a full length 53010 protein or an active fragment thereof.
In a related aspect, the invention further provides nucleic acid constructs that include a 53010 nucleic acid molecule described herein. In certain embodiments, the nucleic acid molecules of the invention are operatively linked to native or heterologous regulatory sequences. Also included, are vectors and host cells containing the 53010 nucleic acid molecules of the invention e.g., vectors and host cells suitable for producing 53010 nucleic acid molecules and polypeptides.
In another related aspect, the invention provides nucleic acid fragments suitable as primers or hybridization probes for the detection of 53010-encoding nucleic acids.
In still another related aspect, isolated nucleic acid molecules that are antisense to a 53010 encoding nucleic acid molecule are provided.
In another aspect, the invention features, 53010 polypeptides, and biologically active or antigenic fragments thereof that are useful, e.g., as reagents or targets in assays applicable to treatment and diagnosis of 53010-mediated or -related disorders. In another embodiment, the invention provides 53010 polypeptides having a 53010 activity. Preferred polypeptides are 53010 proteins including at least one carboxylesterase domain, and, preferably, having a 53010 activity, e.g., a 53010 activity as described herein.
In other embodiments, the invention provides 53010 polypeptides, e.g., a 53010 polypeptide having the amino acid sequence shown in SEQ ID NO:2 or; an amino acid sequence that is substantially identical to the amino acid sequence shown in SEQ ID NO:2; or an amino acid sequence encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under a stringency condition described herein to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, wherein the nucleic acid encodes a full length 53010 protein or an active fragment thereof.
In a related aspect, the invention further provides nucleic acid constructs which include a 53010 nucleic acid molecule described herein.
In a related aspect, the invention provides 53010 polypeptides or fragments operatively linked to non-53010 polypeptides to form fusion proteins.
In another aspect, the invention features antibodies and antigen-binding fragments thereof, that react with, or more preferably specifically bind 53010 polypeptides or fragments thereof, e.g., a carboxylesterase domain.
In another aspect, the invention provides methods of screening for agents, e.g., compounds, that modulate the expression or activity of the 53010 polypeptides or nucleic acids, e.g., compounds that modulate the normal pain response, aberrant or altered pain response, or neurological response.
In a preferred embodiment, the effect of an agent, e.g., a compound, on the pain response is evaluated by an analgesic test, e.g., the hot plate test, tail flick test, writhing test, paw pressure test, all electric stimulation test, tail withdrawal test, or formalin test.
In a preferred embodiment, the agent, e.g., compound, inhibits 53010 activity.
In still another aspect, the invention provides a process for modulating 53010 polypeptide or nucleic acid expression or activity, e.g. using the screened compounds. In certain embodiments, the methods involve treatment of conditions related to aberrant, e.g., decreased or increased expression of the 53010 polypeptides or nucleic acids, such as conditions involving pain response, aberrant or altered pain response, or pain related disorders.
In still another aspect, the invention features a method of modulating (e.g., enhancing or inhibiting) an activity of a cell (e.g., a neural cell), or a response (e.g., a pain response) in a subject. The method includes contacting the cell with, or administered to the subject, an agent, e.g., a compound, that modulates the activity or expression of a 53010 polypeptide or nucleic acid, in an amount effective to modulate the activity or the response.
In a preferred embodiment, the agent modulates (e.g., increases or decreases) carboxylesterase activity.
In a preferred embodiment, the agent modulates (e.g., increases or decreases) expression of the 53010 nucleic acid by, e.g., modulating transcription, mRNA stability, etc.
In a preferred embodiment, the cell, e.g., the 53010-expressing cell, is a central or peripheral nervous system cell, e.g., a cell in an area involved in pain control, e.g., a cell in the brain or spinal cord.
In a preferred embodiment, the agent, e.g., the compound, and the 53010-polypeptide or nucleic acid are contacted in vitro or ex vivo. In a preferred embodiment, the contacting step is effected in vivo in a subject, e.g., as part of a therapeutic or prophylactic protocol. The contacting or administering step(s) can be repeated.
Preferably, the subject is a human, e.g., a patient with pain or a pain-associated disorder disclosed herein. For example, the subject can be a patient with pain elicited from tissue injury, e.g., inflammation, infection, ischemia; pain associated with musculoskeletal disorders, e.g., joint pain; tooth pain; headaches, e.g., migrane; pain associated with surgery; pain related to inflammation, e.g., irritable bowel syndrome; or chest pain. The subject can be a patient with complex regional pain syndrome (CRPS), reflex sympathetic dystrophy (RSD), causalgia, neuralgia, central pain and dysesthesia syndrome, carotidynia, neurogenic pain, refractory cervicobrachial pain syndrome, myofascial pain syndrome, craniomandibular pain dysfunction syndrome, chronic idiopathic pain syndrome, Costen""s pain-dysfunction, acute chest pain syndrome, gynecologic pain syndrome, patellofemoral pain syndrome, anterior knee pain syndrome, recurrent abdominal pain in children, colic, low back pain syndrome, neuropathic pain, phantom pain from amputation, phantom tooth pain, or pain asymbolia. The subject can be a cancer patient, e.g., a patient with brain cancer, bone cancer, or prostate cancer. In other embodiments, the subject is a non-human animal, e.g., an experimental animal, e.g., an arthritic rat model of chronic pain, a chronic constriction injury (CCI) rat model of neuropathic pain, or a rat model of unilateral inflammatory pain by intraplantar injection of Freund""s complete adjuvant (FCA).
In other embodiments, the subject is a human, e.g., a patient with infertility. The subject can be a cancer patient, e.g., a patient with prostate cancer. In yet other embodiments, the subject is a non-human animal, e.g., an experimental animal, e.g., a rodent model for infertility.
In preferred embodiments, the agent is a peptide, a phosphopeptide, a small molecule, e.g., a member of a combinatorial library, or an antibody, or any combination thereof. The antibody can be conjugated to a therapeutic moiety selected from the group consisting of a cytotoxin, a cytotoxic agent and a radioactive metal ion.
In additional preferred embodiments, the agent is an antisense molecule, a ribozyme, a triple helix molecule, or a 53010 nucleic acid, or any combination thereof.
In a preferred embodiment, the agent is administered in combination with a cytotoxic agent.
In another aspect, the invention features a method of treating or preventing, in a subject, a 53010-associated disorder. The method includes administering to the subject, e.g., a subject at risk of, or afflicted with, a 53010-associated disorder, an agent, e.g., a compound as described herein, that modulates the activity or expression of a 53010 polypeptide or nucleic acid, in an amount effective to treat or prevent the disorder.
In a preferred embodiment, the disorder is pain or a pain related disorder.
In a preferred embodiment, the subject is a subject as described herein, e.g., a human.
In still another aspect, the invention features a method for evaluating the efficacy of a treatment of a disorder, e.g., a disorder disclosed herein, in a subject. The method includes treating a subject with a protocol under evaluation; assessing the expression of a 53010 nucleic acid or 53010 polypeptide, such that a change in the level of 53010 nucleic acid or 53010 polypeptide after treatment, relative to the level before treatment, is indicative of the efficacy of the treatment of the disorder.
In a preferred embodiment, the disorder is pain or a pain related disorder.
In a preferred embodiment, the subject is a human.
The invention also features a method of diagnosing a disorder, e.g., a disorder disclosed herein, in a subject. The method includes evaluating the expression or activity of a 53010 nucleic acid or a 53010 polypeptide, such that, a difference in the level of 53010 nucleic acid or 53010 polypeptide relative to a normal subject or a cohort of normal subjects is indicative of the disorder.
In a preferred embodiment, the disorder is pain or a pain related disorder.
In a preferred embodiment, the subject is a human.
In a preferred embodiment, the evaluating step occurs in vitro or ex vivo. For example, a sample, e.g., a blood sample, is obtained from the subject.
In a preferred embodiment, the evaluating step occurs in vivo. For example, by administering to the subject a detectably labeled agent that interacts with the 53010 nucleic acid or polypeptide, such that a signal is generated relative to the level of activity or expression of the 53010 nucleic acid or polypeptide.
The invention also provides assays for determining the activity of or the presence or absence of 53010 polypeptides or nucleic acid molecules in a biological sample, including for disease diagnosis.
In further aspect, the invention provides assays for determining the presence or absence of a genetic alteration in a 53010 polypeptide or nucleic acid molecule, including for disease diagnosis.
In yet another aspect, the invention features a method for identifying an agent, e.g., a compound, which modulates the activity of a 53010 polypeptide, e.g., a 53010 polypeptide as described herein, or the expression of a 53010 nucleic acid, e.g., a 53010 nucleic acid as described herein, including contacting the 53010 polypeptide or nucleic acid with a test agent (e.g., a test compound); and determining the effect of the test compound on the activity of the 53010 polypeptide or nucleic acid to thereby identify a compound which modulates the activity of the 53010 polypeptide or nucleic acid.
In a preferred embodiment, the activity of the 53010 polypeptide is a carboxylesterase activity.
In a preferred embodiment, the activity of the 53010 polypeptide is modulation of pain response.
In preferred embodiments, the agent is a peptide, a phosphopeptide, a small molecule, e.g., a member of a combinatorial library, or an antibody, or any combination thereof.
In additional preferred embodiments, the agent is an antisense, a ribozyme, or a triple helix molecule, or a 53010 nucleic acid, or any combination thereof.
In another aspect, the invention features a two dimensional array having a plurality of addresses, each address of the plurality being positionally distinguishable from each other address of the plurality, and each address of the plurality having a unique capture probe, e.g., a nucleic acid or peptide sequence. At least one address of the plurality has a capture probe that recognizes a 53010 molecule. In one embodiment, the capture probe is a nucleic acid, e.g., a probe complementary to a 53010 nucleic acid sequence. In another embodiment, the capture probe is a polypeptide, e.g., an antibody specific for 53010 polypeptides. Also featured is a method of analyzing a sample by contacting the sample to the aforementioned array and detecting binding of the sample to the array.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.